1. Field of the Invention
This invention relates to a dry-type chemical analytical element used for determination of a particular substance in a body fluid, such as blood.
2. Description of the Prior Art
The quantitative analyses of various metabolic components, such as glucose, bilirubin, urea nitrogen, uric acid, cholesterol, lactate dehydrogenase, creatine kinase, ALT (Alanine aminotransferase) and AST (Aspartate aminotransferase), are important in clinical analysis, particularly in the diagnosis of diseases, the follow-up of the course of treatment, the judgement of prognosis and the like. In clinical assays, where the sample is blood or the like, it is preferable that a highly accurate assay be conducted by using a minute amount of liquid sample. In the past, wet methods using a solution of reagent were widely utilized, however they were poor in rapidity.
On the other hand, dry methods are also known such as clinical assay means. The dry method uses an analytical element, such as, a test piece or a multilayer analytical element, in a substantially dry state where an analytical reagent system is incorporated. The dry methods are superior to the wet methods in terms of simplicity of operation, rapidity, cost, etc. Dry-type multilayer analytical elements have been developed as rapid and accurate assay means, and they are disclosed in U.S. Pat. Nos. 3,992,158, and 4,292,272, EP 0 162 302A, etc. The dry-type multilayer analytical element is, for example, composed of a transparent support, a reagent layer, a light-reflecting layer, a spreading layer, and the like. The transparent support is a subbed thin plastic film or the like. The reagent layer is coated on the support, and contains the reagent which reacts with the analyte in a liquid sample to develop a color, the optical density of which is proportional to the amount of the analyte. The light-reflecting layer functions to block the light incident into the reagent layer so that it does not reach the spreading layer, and to minimize the influence of the liquid sample spotted on the spreading layer at the time of measuring the optical density of the reagent layer. The spreading layer uniformly spreads the liquid sample spotted thereon to an area in proportion to the liquid amount. When quantitative analysis is carried out using the dry-type analytical element, a definite amount of a liquid sample, such as, a whole blood sample is spotted on the spreading layer. The liquid sample spreads in the spreading layer, and passes through the light-reflecting layer. The sample reaches the reagent layer, and reacts with the reagent to form color. After the spotting, the analytical element is incubated for a suitable time at a constant temperature to allow the color reaction to proceed sufficiently. Light is irradiated onto the reagent layer from the side of the transparent support, and reflection optical density is measured at a particular wave length region. The amount of the analyte is determined by using a calibration curve obtained previously.
In the past, the sample to be analyzed was usually blood serum or blood plasma where the erythrocytes were removed, irrespective of whether the wet or dry method was used. However, since the separation of erythrocytes requires labor and equipment cost, analysis is preferably carried out using undiluted whole blood.
When whole blood is analyzed by the dry method, blood cells, i.e. erythrocytes and leukocytes, and other macromolecular components should be separated in the analytical element by some means. For example, the analytical element disclosed in U.S. Pat. No. 3,992,158 is provided with a filtering layer for separating blood cells and other macromolecular components. However, the filtering layer requires a lot of time for the removal of blood cells. Moreover, a part of the analyte is lost in the filtering layer, and thereby, the analysis becomes inaccurate.
Another dry-type analytical element utilizable for the analysis of a particular component in whole blood is disclosed in EP 0 226 465A. In the analytical element, erythrocytes in a whole blood sample are separated from plasma in order to remove the interference of the erythrocytes, and moreover, the analyte in the plasma rapidly diffuses into the reagent layer. The analytical element is composed of a first nonfibrous porous layer, a second nonfibrous porous layer and a fibrous porous layer. They are intergrally and substantially closely laminated in this order each through an adhesive discontinuously disposed so as to form microspaces continuing through from one layer to the next so as not to interfere with the approximately uniform permeation of liquid. The color forming reagent composition is incorporated into any one of the above three porous layers, and the mean effective pore size of the second nonfibrous porous layer is in the range of 0.8 to 30 .mu.m. However, when whole blood samples were analyzed by using the above analytical element, the analytical results among the blood samples having the same analyte content varied considerably depending on the hematocrit values (the volume per cent of blood cells in blood).